Measurements of hydrocortisone and cortisone for longitudinal profiling of equine plasma by liquid chromatography–tandem mass spectrometry

Abstract The conventional detection of exogenous drugs in equine doping samples has been used for confirmation and subsequent prosecution of participants responsible. In recent years, alternative methods using indirect detection have been investigated due to the expanding number of pharmaceutical agents available with the potential of misuse. The monitoring of endogenous biomarkers such as hydrocortisone (HC) has been studied in equine urine with an international threshold of 1 μg/ml established; however, there is no current threshold for equine plasma. The aim of this research was to investigate plasma concentrations of HC and cortisone (C) in race day samples compared to an administration of Triamcinolone Acetonide (TACA). The reference population (n = 1150) provided HC (6 to 145 ng/ml) and C (0.7 to 13 ng/ml) levels to derive the HC to C ratio (HC/C). Population reference limits (PRLs) were proposed for HC/C values at 0.2 (lower) and 61 (upper). Administration of TACA resulted in down‐regulation of HC/C values below the estimated PRLs for up to 96 h post‐administration. This indirect detection period was longer than the detection of TACA for 72 h. The use of individual reference limits (IRLs) for HC/C values was investigated to support the Equine Biological Passport (EBP), an intelligence model developed by Racing NSW for longitudinal monitoring of biomarkers.


SI Section 1: Method Validation Method for Hydrocortisone, Cortisone and TACA quantification
Linearity SI The accuracy of the regression analysis was assessed by analysing the y-residuals from the linearity curve for each of the compounds. This was completed by analysing each replicate of the linearity curve analysing the residual with the expected concentration determined from the linearity and the calculated concentration determined by LabSolutions software. This method is used to account for the differences within the spiking method. To determine if the method is accurate, all points were expected to fall within a range of  1. If the residuals fell outside this range, there would be the presence of bias in the method.

Sensitivity:
Sensitivity was assessed for HC, C and TACA, using a visual comparison of the spikes at a lower concentration in comparison to the plasma calibrators. The LOD and LOQ were estimated by a visual comparison of the smallest peak (usually the tallest noise peak) of the lowest concentration that the instrument could detect for each compound. Using the signal-tonoise ratio (S/N), comparison could be made for a S/N of 3 for LOD and a S/N of 10 for LOQ. Concentrations of 0.05, 0.1, 0.2 and 0.5 ng/mL were chosen for HC and C whilst for TACA, concentrations of 0.01, 0.02, 0.03, 0.04, 0.05 and 0.1 ng/mL.

Accuracy:
Accuracy was estimated by the calculation of the precent relative error (%RE) from the mean concentration of each QC sample compared to the theoretical concentration. Each QC was completed in duplicate.

Precision:
Precision was estimated as %RSD using the average concentrations of duplicate QC samples. Precision was deemed to be acceptable within < 20%.

Recovery:
Recovery was assessed by comparing pre-and post-SPE equine plasma spike replicates (n=7) at the QC levels with recovery expected to be higher than 50%.

Matrix Effects:
Matrix effects were assessed by comparing post-SPE spiked plasma samples to neat standards (n=7). Matrix effects greater than 100%, indicated ion enhancement, while less than 100% indicated ion suppression.

Dilution Factor:
Dilution factor was assessed using concentrations of 7 replicated samples that represented either a 1 mL or 0.5 mL of solution. The chosen concentration of HC for the spike was 300 ng/mL as dilution for 1 mL (1 in 2) and 0.5 mL (1 in 4) sample volume would fall within the chosen calibration range. The concentration determined by the instrument was then multiplied by the relevant dilution factor to obtain the sample concentration. For the dilution to be deemed acceptable, this required a %RE of ≤ 15%.

Stability:
Stability was assessed over a 3-month period at two different temperatures: 4°C and -20°C in the DCM: EtOH surrogate matrix. Each QC sample was spiked in duplicate with the analyte considered stable if the concentration was within ± 20 ng/mL.

SI Section 2: Results and Discussion:
SI

SI Section 3: Surrogate Matrix Optimisation:
A surrogate matrix was used as the y-intercept for HC using standard addition for the calibration range was very high (greater than 6). This high y-intercept is not considered suitable as this study required an accurate method to determine low levels of HC as the TACA administration resulted in down-regulation of HC. So, whilst the calibration range using standard addition was acceptable, the high y-intercept wasn't therefore the need for a surrogate matrix for an accurate calibration was necessary.
Various methods were explored to optimise the surrogate matrix including solid phase extraction (SPE) and liquid-liquid extraction (LLE). Blank plasma from a pooled collection of plasma known to not contain any exogenous drugs was used to produce the surrogate matrix. For LLE, 3 mL of blank plasma was transferred from the pooled amount into screw top test tubes, 4 mL of di-isopropyl ether (DIPE) was added to each tube and rotated on the mixer for 20 minutes at medium speed allowing for complete inversion between the plasma and the DIPE. Each tube was further centrifuged for 3000 rpm for 10 minutes then the aqueous plasma layer from each tube was extracted into a clear plastic bottle and stored at 4 °C until use. This process was completed a total of 3 times for each bottle of stripped plasma that underwent LLE using DIPE.
Another 2 sets of LLEs were completed using either dichloromethane (DCM) and methanol (MeOH) in a 90:10 ratio or dichloromethane (DCM) and ethanol (EtOH) in a 90:10 ratio. For each of these mixtures, 3 mL of blank plasma was transferred from the pooled amount into screw top test tubes. Into each test tube, 3 mL of either DCM:MeOH or DCM:EtOH was added, rotated on a mixer for 20 minutes at medium speed to allow for complete inversion between the plasma and the extraction solution. Each tube was centrifuged at 3000 rpm for 10 minutes then the aqueous plasma layer from each tube was extracted into a clear plastic bottle and then stored at 4 °C until use.
For SPE, a clean-up carbon extraction cartridge from UCT (Bristol, PA, United States of America) was used. Cartridges were pre-conditioned with approximately 3 mL of purified water, 3 mL of blank plasma from the pooled amount was then transferred into each carbon cartridge. Plasma was allowed to run through the carbon cartridge using gravity to filter the plasma through the cartridge into a test tube.
Using HC as an example, LLE using three lots of DIPE removed 12%, SPE carbon cartridges removed 30% whilst LLE DCM:MeOH in a 90:10 ratio removed 90% of endogenous HC. The best method to produce the surrogate matrix utilised DCM:EtOH in a 90:10 ratio, which successfully removed 99% of the endogenous HC (SI Figure S11). The use of DCM:EtOH in a 90:10 ratio also allowed for the y-intercept to be reduced from originally 6.99 to 0.15 therefore allowing for a more accurate calibration range to detect lower levels of HC. This result was consistent with previous studies conducted by Popot et al. for equine urine which is advantageous as this method was used to establish the hydrocortisone threshold in urine. Figure S11: Amount of hydrocortisone removed from blank plasma using DCM:EtOH (90:10 v/v)